Identification of two separate allelic mutations in the lipoprotein lipase gene of a patient with the familial hyperchylomicronemia syndrome.

The molecular defects resulting in a deficiency of lipoprotein lipase activity in a patient with the familial hyperchylomicronemia syndrome have been identified. Increased lipoprotein lipase mass but undetectable lipoprotein lipase activity in the patient's post-heparin plasma indicate the presence of an inactive enzyme. No major gene rearrangements were identified by Southern blot analysis of the patient's lipoprotein lipase gene and Northern blot hybridization revealed an lipoprotein lipase mRNA of normal size. Sequence analysis of polymerase chain reaction-amplified lipoprotein lipase cDNA identified two separate allelic mutations. A T to C transition at nucleotide 836 results in the substitution of Ile194, located near the putative interfacial recognition site of lipoprotein lipase, to a Thr. A G to A mutation at base 983 leads to the substitution of a His for Arg243 and the loss of a HhaI restriction enzyme site. Arg243 is near His241, which has been postulated to be part of the catalytic triad of lipoprotein lipase. Direct sequencing of amplified cDNA and digestion with HhaI established that the proband is a compound heterozygote for each base substitution. Transient expression of each of the mutant lipoprotein lipase cDNAs in human embryonal kidney-293 cells resulted in the synthesis of enzymically inactive proteins, establishing the functional significance of the mutations. We conclude that the Ile194 to Thr194 and Arg243 to His243 substitutions occur in lipoprotein lipase regions essential for normal enzyme activity and each mutation results in the expression of a nonfunctional enzyme leading to the hyperchylomicronemia syndrome manifested in the proband.     

Synthesis of inactive nonsecretable high mannose-type lipoprotein lipase by cultured brown adipocytes of combined lipase-deficient cld/cld mice

Combined lipase deficiency (cld) is a recessive mutation which causes a severe deficiency of lipoprotein lipase and hepatic lipase activities and lethal hypertriacylglycerolemia within 3 days in newborn mice. The effect of this genetic defect on lipoprotein lipase was studied in primary cultures of brown adipocytes derived from tissue of newborn mice. Cells cultured from cld/cld mice replicated, accumulated triacylglycerol, and differentiated into adipocytes at normal rates. Lipoprotein lipase activity in unaffected cells was detectable on Day 0 of confluence and increased to 1.3 units/mg DNA by Day 6, while that in cld/cld cells was less than 4% of that in unaffected cells on Days 4-6. Unaffected cells released 1.2% of their lipase activity in 30 min in the absence of heparin, and 11% in 10 min in the presence of heparin, whereas cld/cld cells released no lipase activity. cld/cld cells contained 2-3 times as much lipoprotein lipase protein as unaffected cells, and released no lipase protein to the medium. Immunofluorescent lipoprotein lipase was not detectable in unaffected adipocytes unless lipase secretion was blocked with monesin, causing retention of the lipase in Golgi. cld/cld adipocytes, in contrast, contained immunofluorescent lipoprotein lipase distributed in a diffuse reticular pattern, indicating retention of lipase in endoplasmic reticulum. Lipoprotein lipase immunoprecipitated from cells incubated 1-3 h with [35S]methionine was digested with or without endoglycosidase H (endo H) or F, and resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lipoprotein lipase in unaffected cells (Mr = 56,000-58,000) consisted of three glycosylated forms, of which the most prevalent was endo H-resistant, the next was totally endo H-sensitive, and the least was partially endo H-sensitive. In contrast, lipoprotein lipase in cld/cld cells (Mr = 56,000) consisted of a single, totally endo H-sensitive form. Lipoprotein lipase in both groups of cells contained two oligosaccharide chains. Chromatography studies with heparin-Sepharose indicated that at least some of the lipoprotein lipase in cld/cld cells was dimerized. The findings demonstrate that brown adipocytes cultured from cld/cld mice synthesize lipoprotein lipase with two high mannose oligosaccharide chains, but it is inactive and retained in endoplasmic reticulum. Whether the cld mutation affects primarily processing of oligosaccharide chains of lipoprotein lipase in endoplasmic reticulum, transport of the lipase from the reticulum, or some other process, is to be resolved.     

Partial gene duplication involving exon-Alu interchange results in lipoprotein lipase deficiency.

Major structural rearrangements are uncommon causes of mutation in human genetic diseases. We have previously described that a significant proportion of unrelated patients of western European descent who are deficient in lipoprotein lipase (LPL) activity have a major structural rearrangement in the LPL gene. Here we report the detailed characterization of this mutation. We show that this rearrangement is due to a duplication of approximately 2 kb which results from juxtaposition of intron 6 to a partially duplicated exon 6. We have sequenced both the junction fragment of this duplication and the corresponding wild-type regions and have found that the breakpoint in intron 6 is associated with the simple repeat found at the 3' end of an Alu element. The breakpoint within exon 6 shows no homology to this simple repeat. This result both suggests that this interchange arose as a nonhomologous recombination event and shows that such events resulting in duplication which occur in normal gene evolution may also lead to genetic disease. Cloning of the junction fragment has allowed synthesis of appropriate primers for rapid screening for this rearrangement in other families with LPL deficiency.     

Combined lipase deficiency (cld/cld) in mice. Demonstration that an inactive form of lipoprotein lipase is synthesized

Combined lipase deficiency, cld, is a recessive mutation within the T/t complex of mouse chromosome 17. Mice homozygous for this defect display severe functional deficiencies of lipoprotein lipase and the related hepatic lipase. They develop massive hyperchylomicronemia and die within 3 days when allowed to suckle. Heart, diaphragm muscle, and brown adipose tissue of 1-day-old cld/cld and unaffected mice incorporated in vivo [35S]methionine into a protein that could be immunoprecipitated by antilipoprotein lipase serum. The immunoprecipitated protein in all tissues had the same Mr as bovine lipoprotein lipase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proportion of radioactivity in the lipoprotein lipase band to that in total protein was 0.02% in tissues of cld/cld mice and 0.01% in tissues of unaffected mice. There was 2-6 times more lipoprotein lipase-like protein (determined by immunoassay) in tissues of defective mice than in those of unaffected mice. These findings indicate that the cld mutation did not cause deletion of the structural gene for lipoprotein lipase. Lipoprotein lipase activity in heart, diaphragm muscle, brown adipose tissue, and lung of cld/cld mice was less than 5% of that in tissues of unaffected mice. This low activity could be inhibited more than 85% by antilipoprotein lipase serum, but not by nonimmune serum. It is concluded that tissues in cld/cld mice synthesize a lipoprotein lipase-like protein which has subnormal catalytic activity.     

Nature and frequency of mutations in the argininosuccinate synthetase gene that cause classical citrullinemia

Citrullinemia is an autosomal recessive disorder caused by a genetic deficiency of argininosuccinate synthetase (ASS). So far 20 mutations in ASS mRNA have been identified in human classical citrullinemia, including 14 single base changes causing missense mutations in the coding sequence of the enzyme, 4 mutations associated with an absence of exons 5, 6, 7, or 13 in mRNA, 1 mutation with a deletion of the first 7 bases in exon 16 (which is caused by abnormal splicing), and 1 mutation with an insertion of 37 bases between the exon 15 and 16 regions in mRNA. In order to identify the abnormality in the ASS gene causing the exon 7 and 13 deletion mutations and the 37-base insertion mutation between exons 15 and 16 in mRNA, and to establish a DNA diagnostic test, we isolated and sequenced the genomic DNA surrounding each exon. The absence of exon 7 or 13 in ASS mRNA resulted from abnormal splicing caused by a single base change in the intron region: IVS-6^–2 (a transition of A to G at the second nucleotide position within the 3 splice cleavage site of intron 6) and IVS-13⁺⁵ (a transition of G to A at the fifth nucleotide position within the 5 splice cleavage site of intron 13), respectively. The IVS-6^–2 mutation resulted in the creation of an MspI restriction site. DNA diagnostic analysis of 33 Japanese alleles with classical citrullinemia showed that 19 alleles had the IVS-6^–2 mutation (over 50% of the mutated alleles in Japanese patients). It was thus confirmed that one mutation is predominant in Japan. This differs from the situation in the USA where there is far greater heterogeneity. The insertion mutation in mRNA on the other hand resulted from abnormal splicing caused by a 13-bp deletion at the splice-junction between exon 15 and intron 15. The deletion had a short direct repeat (CTCAGG) at the breakpoint junction and presumably resulted from slipped mispairing.     

Changes in the expression of uncoupling proteins and lipases in porcine adipose tissue and skeletal muscle during feed deprivation*(1)

The hormone-sensitive and lipoprotein lipases are critical determinants of the metabolic adaptation to starvation. Additionally, the uncoupling proteins have emerged with potential roles in the metabolic adaptations required by energy deficiency. The objective of this study was to evaluate the expression (mRNA abundance) of uncoupling proteins 2 and 3 and that of hormone-sensitive and lipoprotein lipase in the adipose tissue and skeletal muscle of the pig in relationship to feed deprivation. Thirty-two male castrates (87 kg +/- 5%) were assigned at random to fed and feed-deprived treatment groups. After 96 hr, the pigs were euthanized and adipose and skeletal muscle tissue obtained for total RNA extraction and nuclease protection assays. Feed deprivation increased uncoupling protein 3 mRNA abundance 103-237% (P < 0.01) in longissimus and red and white semitendinosus muscle. In contrast, the increase in uncoupling protein 3 mRNA in adipose tissue was only 23% (P < 0.06), and adipose uncoupling protein 2 mRNA was not influenced (P > 0.66) by feed deprivation. The increased abundance of uncoupling protein 2 mRNA in the longissimus muscle of feed-deprived pigs was small (22%), but significant (P < 0.04). The expression of hormone-sensitive lipase was increased 46% and 64% (P < 0.04) in adipose tissue and longissimus muscle, respectively, by feed deprivation, whereas adipose lipoprotein lipase expression was reduced (P < 0.01) to 20% of that of the fed group. Longissimus lipoprotein lipase expression in the feed-deprived group was 37% of that of the fed group (P < 0.01), and similar reductions were detected in red and white semitendinosus muscle. Overall, these findings indicate that uncoupling protein 3 expression in skeletal muscle is quite sensitive to starvation in the pig, whereas uncoupling protein 2 changes are minimal. Furthermore, we conclude that hormone-sensitive lipase is upregulated at the mRNA level with prolonged feed deprivation, whereas lipoprotein lipase is downregulated.     

Deletion of exon encoding cysteine-rich repeat of low density lipoprotein receptor alters its binding specificity in a subject with familial hypercholesterolemia

The proposed ligand binding domain of the low density lipoprotein (LDL) receptor consists of a 40-amino acid cysteine-rich unit that is repeated with some variation seven times. We describe here a mutant allele at the LDL receptor locus in which one of the seven repeats has been deleted. This mutation was found in a patient with the clinical syndrome of homozygous familial hypercholesterolemia. By molecular cloning, we show that the deletion arose by homologous recombination between repetitive Alu sequences in intron 4 and intron 5 of the gene. The deletion removes exon 5, which normally encodes the sixth repeat of the ligand binding domain. In the resultant mRNA, exon 4 is spliced to exon 6, preserving the reading frame. This mRNA produces a shortened protein that reaches the cell surface and reacts with anti-receptor antibodies but does not bind LDL, which contains apoprotein B-100 as its major protein component. Surprisingly, the deleted protein retains the ability to bind and internalize beta-migrating very low density lipoprotein, a lipoprotein that contains apoprotein E as well as apoprotein B-100. These data support the hypothesis that the seven repeated sequences in the receptor constitute the LDL binding domain. The data further indicate that the sixth repeat is required for binding of LDL, but not beta-migrating very low density lipoprotein, and that deletion of a single cysteine-rich repeat can alter the binding specificity of the LDL receptor.     

Tissue-specific alterations in lipoprotein lipase activity in copper-deficient rats

Copper deficiency results in alterations in lipid metabolism that include elevations in serum cholesterol and triglycerides and a decrease in whole-body respiratory quotient. Copper-deficient animals are also leaner even though electron micrographs of the myocardium present increased lipid droplet accumulation. To address whether a compromised copper status impacts triglyceride deposition in a tissue-specific manner, the activity of lipoprotein lipase was measured in adipose tissue and cardiac and skeletal muscle. Weanling rats fed a copper-restricted diet (<1 ppm) for 6 wk demonstrated a greater than twofold increase in cardiac lipoprotein lipase activity concomitant with a significant reduction in adipose tissue lipoprotein lipase activity. Skeletal muscle lipoprotein lipase activity was not altered by the copper-deficient state. The results of this study suggest that copper deficiency may induce a tissue-specific alteration in lipoprotein lipase activity in rats, which may contribute to the notable deposition of lipid substance in myocardium and the concomitant general body leanness.     

Restoration of lipoprotein lipase activity in insulin-deficient rats by insulin infusion is tissue-specific

The activity of lipoprotein lipase was measured in white and brown adipose tissues, red vastus lateralis muscle, and heart of rats that have been insulin deficient (streptozotocin, 75 mg.kg-1) for 2 weeks, and that have then received implants of insulin-delivering minipumps (17 U.kg-1.day-1) for 1 or 4 days. Normal glycemia was restored in insulin-deficient animals after 4 days of insulin treatment. Hypertriglyceridemia, but not hypercholesterolemia, was reversed after 4 days of insulin infusion. After 2 weeks of insulin deficiency, fasting lipoprotein lipase activity was lowered in all tissues studied. In white adipose tissue, lipoprotein lipase decreased to 50% of control values. After a single day of insulin infusion, even if tissue weight has not yet been greatly affected, total activity was completely restored to control levels. Enzyme activity in brown adipose tissue was also depressed in deficient animals, and insulin infusion was followed by a slow recovery of activity, to a level intermediate between those of control and insulin-deficient groups. Insulin status had milder effects on lipoprotein lipase activity in vastus lateralis muscle than in the adipose tissues. Deficient rats displayed 60% less activity than controls, and 4 days of hormone infusion only partially restored enzyme activity. There was a large loss of lipoprotein lipase in the heart following 2 weeks of insulin depletion, which was not counteracted by hormone infusion. Thus the speed and extent of recovery of lipoprotein lipase activity following hormone replacement in insulin-deficient animals varied widely among tissues. These findings suggest that insulin is part of the factors that determine the tissue specificity of lipoprotein lipase regulation.     

Lipoprotein lipase genotypes for a common premature termination codon mutation detected by PCR-mediated site-directed mutagenesis and restriction digestion.

We have developed a procedure for the determination of a common mutation in exon 9 of the human lipoprotein lipase (LPL) gene. The mutation is due to a C-G transversion which creates a premature termination codon (Ser447-Ter) and results in a truncated LPL molecule lacking the C-terminal dipeptide SER-GLY. The mutation can be detected by polymerase chain reaction (PCR) amplification of exon 9 using a modified 3' amplimer that produces a 140 bp product containing a site for the restriction enzyme Hinf-1 in the presence of the mutation (G allele). The G allele was in strong linkage disequilibrium with a Hind-III restriction fragment length polymorphism (RFLP) allele in intron 8. Genotype determinations for the mutation can be performed by PCR amplification of genomic DNA, digestion with Hinf-1, and analysis of the products by polyacrylamide gel electrophoresis. The allelic frequency of the Ser447-Ter mutation in normal male Caucasian controls was 0.11. The frequency of the mutation was lower in a group of subjects with primary hypertriglyceridemia compared to normolipidemic controls.     

Avian adipose lipoprotein lipase: cDNA sequence and reciprocal regulation of mRNA levels in adipose and heart

cDNA clones for chicken adipose lipoprotein lipase were isolated from an expression library in lambda gt11 by antibody screening and characterized by hybridization selection and nucleotide sequencing. Based on the cDNA sequence and on N-terminal sequence analysis of the purified enzyme, chicken adipose lipoprotein lipase is a mature protein of 465 amino acids with a signal peptide of 19 or 25 amino acids, depending on which of two methionine residues is used for translation initiation. The predicted amino-acid sequence was found to be 73-77% identical to the four known mammalian adipose lipoprotein lipase sequences, with conservation of position of cysteine residues and putative functional domains, and number of potential N-glycosylation sites. Chicken lipoprotein lipase differs from mammalian lipoprotein lipases with respect to the position of one N-glycosylation site and the presence of an additional 15-17 C-terminal amino acids. 32P-labeled cDNA clones hybridized to mRNA species of 3.7 and 4.0 kb in Northern blots of heart and adipose, but not of liver RNA. In chickens that were fasted for 48 h and then refed, lipoprotein lipase mRNA levels in adipose increased to a maximal level of 350% that of controls at 10 h, whereas heart lipoprotein lipase mRNA levels fell to 40% of controls at 14 h. Concomitantly, no changes in total RNA were observed. Thus, avian lipoprotein lipase is subject to reciprocal pretranslational regulation in adipose and heart.     

The molecular basis of a familial apoE deficiency. An acceptor splice site mutation in the third intron of the deficient apoE gene

The molecular basis of the familial apoE deficiency was investigated by gene cloning and comparative expression studies of the normal and the deficient apoE gene. For the latter studies the apoE genes were placed under the control of the mouse metallothionein I promoter in a bovine papilloma virus vector. The studies showed that in the normal gene the mouse metallothionein I promoter directs the synthesis of normal apoE mRNA and protein. In contrast, in the deficient apoE gene the same promoter directs the synthesis of two abnormal apoE mRNA forms, which are similar to those observed in the peripheral blood monocyte macrophage cultures of the patient. Restriction analysis of the cloned gene and partial DNA sequence has shown an A to G substitution in the penultimate 3' nucleotide of the third intron of the apoE gene. This single base substitution abolishes the correct 3' splice site thus creating two abnormally spliced mRNA forms. The smaller form contains 53 nucleotides and the larger form contains the entire third intron of the apoE gene. Both of these mRNA species contain chain termination codons within the intronic sequence and code for short apoE peptides that are not detectable by gel electrophoretic techniques. These findings show that this form of familial apoE deficiency results from a point mutation in the 3' splice junction of the third intron of the apoE gene. Furthermore, the expression system we have employed to study apoE deficiency can be utilized to analyze a broad spectrum of genetic diseases associated with mRNA processing mutations.     

Hypertriglyceridemia in lecithin-cholesterol acyltransferase-deficient mice is associated with hepatic overproduction of triglycerides, increased lipogenesis, and improved glucose tolerance

Lecithin-cholesterol acyltransferase deficiency is frequently associated with hypertriglyceridemia (HTG) in animal models and humans. We investigated the mechanism of HTG in the ldlr-/- x lcat-/- (double knockout (dko)) mice using the ldlr-/- x lcat+/+ (knock-out (ko)) littermates as control. Mean fasting triglyceride (TG) levels in the dko mice were elevated 1.75-fold compared with their controls (p < 0.002). Both the very low density lipoprotein and the low density lipoprotein/intermediate density lipoprotein fractions separated by fast protein liquid chromatography were TG-enriched in the dko mice. In vitro lipolysis assay revealed that the dko mouse very low density lipoprotein (d < 1.019 g/ml) fraction separated by ultracentrifugation was a more efficient substrate for lipolysis by exogenous bovine lipoprotein lipase. Post-heparin lipoprotein lipase activity was reduced by 61% in the dko mice. Hepatic TG production rate, determined after intravenous Triton WR1339 injection, was increased 8-fold in the dko mice. Hepatic mRNA levels of sterol regulatory element binding protein-1 (srebp-1) and its target genes acetyl-CoA carboxylase-1 (acc-1), fatty acid synthase (fas), and stearoyl-CoA desaturase-1 (scd-1) were significantly elevated in the dko mice compared with the ko control. The hepatic mRNA levels of LXRalpha (lxralpha) and its target genes including angiopoietin-like protein 3 (angptl-3) in the dko mice were unchanged. Fasting glucose and insulin levels were reduced by 31 and 42%, respectively in the dko mice, in conjunction with a 49% reduction in hepatic pepck-1 mRNA (p = 0.014). Both the HTG and the improved fasting glucose phenotype seen in the dko mice are at least in part attributable to an up-regulation of the hepatic srebp-1c gene.     

Characterization of a new splicing mutation in the steroid 21-hydroxylase gene

We identified a novel mutation in the CYP21A2 gene, a C to G substitution in the 7-position of the intron 2 acceptor splice site (c.290-7C>G), which causes a steroid 21-hydroxylase deficiency. The effect of the mutation on splicing was checked in the system of CYP21A minigene expression in cultured mammalian cells. The mutation impairs the use of the intron 2 acceptor splice site, resulting in intron retention in mRNA.     

Missense mutation (Gly----Glu188) of human lipoprotein lipase imparting functional deficiency

Cloning and sequencing of lipoprotein lipase (LPL) cDNA prepared from the adipose tissue of a patient with classical LPL deficiency revealed a G to A transition at nucleotide 818 in all sequenced clones, leading to the substitution of glutamic acid for glycine at residue 188 of the mature protein. Hybridization of genomic DNA with allele-specific oligonucleotides confirmed that the patient was homozygous for this mutation and revealed that carrier status for this mutation among relatives of the patient was significantly associated with hypertriglyceridemia. Assay of the patient's plasma for immunoreactive enzyme and activity demonstrated the presence of a circulating inactive enzyme protein, the concentration of which was further increased by injection of heparin. The mutant sequence was produced by oligonucleotide-directed mutagenesis, and both normal and mutant sequences were cloned into the expression vector pSVL and transfected into COS-1 cells. The normal sequence led to the in vitro expression of an enzyme that bound to heparin-Sepharose and had a specific catalytic activity similar to that of normal postheparin plasma enzyme. By contrast, the mutant enzyme expressed in vitro was catalytically inactive and displayed a lower affinity for heparin than the normal enzyme. We conclude that this single amino acid substitution leads to the in vivo expression of an inactive enzyme accounting for the manifestations of LPL deficiency noted in the patient.